Within the United States, ongoing research is directed toward developing alternative energy sources to reduce our dependence on foreign oil and nonrenewable energy. The use of ethanol as a fuel has become increasingly prevalent in recent years. Currently, corn is the primary carbon source used in ethanol production. The use of corn in ethanol production is not economically sustainable and, arguably, may result in increased food costs.
In order to meet the increased demand for ethanol, it will be necessary to ferment sugars from other biomass, such as agricultural wastes, corn hulls, corncobs, cellulosic materials, pulping wastes, fast-growing hardwood species, and the like. Biomass from most of these sources contains large amounts of xylose, constituting about 20 to 25% of the total dry weight. Because agricultural residues have a high xylose content, the potential economic and ecologic benefits of converting xylose in these renewable materials to ethanol are significant.
In biomass conversion, microorganisms serve as biocatalysts to convert cellulosic materials into usable end products such as ethanol. Efficient biomass conversion in large-scale industrial applications requires a microorganism that can tolerate high sugar and ethanol concentrations, and which is able to ferment multiple sugars simultaneously.
The pentose D-xylose is significantly more difficult to ferment than D-glucose. Bacteria can ferment pentoses to ethanol and other co-products, and bacteria with improved ethanol production from pentose sugars have been genetically engineered. However, these bacteria are sensitive to low pH and high concentrations of ethanol, their use in fermentations is associated with co-product formation, and the level of ethanol produced remains too low to make the use of these bacteria in large-scale ethanol production economically feasible.
In general, industrial producers of ethanol strongly favor using yeast as biocatalysts, because yeast fermentations are relatively resistant to contamination, are relatively insensitive to low pH and ethanol, and are easier to handle in large-scale processing. Many different yeast species use xylose respiratively, but only a few species use xylose fermentatively. Fermentation of xylose to ethanol by wild type xylose-fermenting yeast species occurs slowly and results in low yields, relative to fermentation rates and ethanol yields that are obtained with conventional yeasts in glucose fermentations. In order to improve the cost effectiveness of xylose fermentation, it is necessary to increase the rate of fermentation and the ethanol yields obtained.
The most commonly used yeast in industrial applications is Saccharomyces cerevisiae. Although S. cerevisiae is unable to grow on or ferment xylose, homogenates of S. cerevisiae can readily ferment D-ribulose-5-phosphate to ethanol, and can convert D-xylulose-5-phosphate to a lesser extent. S. cerevisiae metabolically engineered to overproduce D-xylose reductase (XYL1), xylitol dehydrogenase (XYL2) and D-xylulokinase (XYL3 or XKS1) or some forms of xylose isomerase (xylA) along with XYL3 or XKS1 can metabolize xylose to ethanol.
Pichia stipitis is a yeast species that in its native state is able to ferment xylose to produce ethanol. In P. stipitis, fermentative and respirative metabolism co-exist to support cell growth and the conversion of sugar to ethanol.
There is a need in the art for yeast strains having improved ability to convert sugars to ethanol.